A high performance liquid chromatographic (HPLC) method for rapid simultaneous determination of alanine-tyrosine (AY) and 20 amino acids in corn oligopeptides was performed automatically by the autosampler, based on the online derivatization of amino groups with o-phthaldialdehyde (OPA) and 9-fluorenylmethyl chloroformate (FMOC-Cl) and the detection was carried out with fluorometric detection combined a C-18 column using gradient elution with external standard. The separation of 20 amino acids and AY was achieved within 30 min on a reversed-phase column (Agilent Hypersil ODS column, 125 mm×4.6 mm×5 µm) with fluorometric detection (excitation wavelength at 340 nm and emission wavelength at 450 nm) by elution buffers based on sodium acetate with the flow rate 1.0 mL/min. The method linearity, calculated for each amino acid and AY, has a correlation coefficient higher than 0.990, in concentrations ranging from 0.1 to 1000.0 mg/L. The stability of derivatives in acidified samples at 4°C and room temperature was demonstrated. The limit of quantitation was estimated to be varying between 0.005 mg/L (AY) and 0.02 mg/L(Asp) and recovery rates between 96.3% (Cys) and 102.8% (AY). The repeatability of the method, expressed as R.S.D., ranged from 1.06 to 4.51%. The method employs protein precipitation with trichloroacetic acid (TCA) and selected the reaction coil as derivatization. TCA clean-up showed a sufficient recovery for peptides as direct injection of the supernatant without evaporation or dilution. The presented method was applied for the quantitation of amino acid and AY contents in corn oligopeptides producing from microbial fermentation, complex enzyme hydrolysis and single enzyme hydrolysis methods.
Online ISSN: 1459-0263Year: 2013, Vol. 11, Issue 1, pages 86-90. Publisher: WFL.
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